Processes for the formulation of pneumococcal polysaccharides for conjugation to a carrier protein

ABSTRACT

The present invention provides a number of process improvements related to the conjugation of capsular polysaccharides from  Streptococcus pneumoniae  to a carrier protein. These process are serotype specific and include acid hydrolysis, addition of sodium chloride to the reductive amination reaction, and addition of sucrose to dissolve polysaccharides. Polysaccharide-protein conjugates prepared using the processes of the invention can be included in multivalent pneumococcal conjugate vaccines.

FIELD OF INVENTION

The present invention provides a number of process improvements relatedto the conjugation of capsular polysaccharides from Streptococcuspneumoniae to a carrier protein. Polysaccharide-protein conjugatesprepared using the processes of the invention can be included inmultivalent pneumococcal conjugate vaccines.

BACKGROUND OF THE INVENTION

Streptococcus pneumoniae, one example of an encapsulated bacterium, is asignificant cause of serious disease world-wide. In 1997, the Centersfor Disease Control and Prevention (CDC) estimated there were 3,000cases of pneumococcal meningitis, 50,000 cases of pneumococcalbacteremia, 7,000,000 cases of pneumococcal otitis media and 500,000cases of pneumococcal pneumonia annually in the United States. SeeCenters for Disease Control and Prevention, MMWR Morb Mortal Wkly Rep1997, 46(RR-8):1-13. Furthermore, the complications of these diseasescan be significant with some studies reporting up to 8% mortality and25% neurologic sequelae with pneumococcal meningitis. See Arditi et al.,1998, Pediatrics 102:1087-97.

The multivalent pneumococcal polysaccharide vaccines that have beenlicensed for many years have proved invaluable in preventingpneumococcal disease in adults, particularly, the elderly and those athigh-risk. However, infants and young children respond poorly tounconjugated pneumococcal polysaccharides. Bacterial polysaccharides areT-cell-independent immunogens, eliciting weak or no response in infants.Chemical conjugation of a bacterial polysaccharide immunogen to acarrier protein converts the immune response to a T-cell-dependent onein infants. Diphtheria toxoid (DTx, a chemically detoxified version ofDT) and CRM197 have been described as carrier proteins for bacterialpolysaccharide immunogens due to the presence of T-cell-stimulatingepitopes in their amino acid sequences.

The pneumococcal conjugate vaccine, Prevnar®, containing the 7 mostfrequently isolated serotypes (4, 6B, 9V, 14, 18C, 19F and 23F) causinginvasive pneumococcal disease in young children and infants at the time,was first licensed in the United States in February 2000. Followinguniversal use of Prevnar® in the United States, there has been asignificant reduction in invasive pneumococcal disease in children dueto the serotypes present in Prevnar®. See Centers for Disease Controland Prevention, MMWR Morb Mortal Wkly Rep 2005, 54(36):893-7. However,there are limitations in serotype coverage with Prevnar® in certainregions of the world and some evidence of certain emerging serotypes inthe United States (for example, 19A and others). See O'Brien et al.,2004, Am J Epidemiol 159:634-44; Whitney et al., 2003, N Engl J Med348:1737-46; Kyaw et al., 2006, N Engl J Med 354:1455-63; Hicks et al.,2007, J Infect Dis 196:1346-54; Traore et al., 2009, Clin Infect Dis48:S181-S189.

Prevnar 13® is a 13-valent pneumococcal polysaccharide-protein conjugatevaccine including serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A,19F and 23F. See, e.g., U.S. Patent Application Publication No. US2006/0228380 A1, Prymula et al., 2006, Lancet 367:740-48 and Kieningeret al., Safety and Immunologic Non-inferiority of 13-valent PneumococcalConjugate Vaccine Compared to 7-valent Pneumococcal Conjugate VaccineGiven as a 4-Dose Series in Healthy Infants and Toddlers, presented atthe 48^(th) Annual ICAAC/ISDA 46^(th) Annual Meeting, Washington D.C.,Oct. 25-28, 2008. See, also, Dagan et al., 1998, Infect Immun. 66:2093-2098 and Fattom, 1999, Vaccine 17:126.

The current multivalent pneumococcal conjugate vaccines have beeneffective in reducing the incidence of pneumococcal disease associatedwith those serotypes present in the vaccines. However, the prevalence ofthe pneumococci expressing serotypes not present in the vaccine has beenincreasing. The process conditions for novel serotypes has to bedetermined for each serotype for conjugation efficiency and for certainserotypes presented unique challenges. Accordingly, there is a need forimproved process conditions for conjugating novel pneumococcal serotypesfor inclusion in future vaccines.

SUMMARY OF THE INVENTION

The present invention provides a number of process changes in thepreparation of polysaccharides (Ps) from Streptococcus pneumoniae thatare unique to specific serotypes. These process changes improve theproperties of the polysaccharide and/or the polysaccharide dissolution,resulting in better conjugation.

In one embodiment, the invention provides process conditions forobtaining S. pneumoniae polysaccharides from serotypes 12F, 23A, 24F,and 31 of a reduced size, which when conjugated to a carrier protein(Pr) in an aprotic solvent show desired conjugate attributes.Specifically, Ps size reduction of these serotypes by acid hydrolysisyields lower Ps molecular mass for protein conjugation compared tohomogenization, which improves conjugate attributes such as lysineconsumption, free Ps or free Pr.

In one embodiment, the invention provides process conditions forobtaining improved polysaccharide-protein conjugate attributes afterconjugation in an aprotic solvent such as DMSO using sodium chloride,particularly for S. pneumoniae polysaccharides from serotypes 15A, 16F,17F, 20, 24F, and 35B. Specifically, in this embodiment, inclusion of ≥1mM sodium chloride prior to or during the conjugation reaction(regardless of where in the process the sodium chloride is added)results in improved conjugate attributes such as larger conjugate size,higher lysine consumption, lower free Ps or free Pr.

In one embodiment, the invention provides a range of pre-lyophilizationformulation conditions for S. pneumoniae polysaccharides of serotypes 3,8, and 24F to ensure complete dissolution following lyophilization.Specifically, polysaccharides are formulated with sucrose and water,such that the sucrose to polysaccharide mass ratio is ≥30×, andoptimally ≥40×. For example, for a given pre-lyophilizationpolysaccharide concentration of 2 mg Ps/mL, sucrose concentration shouldminimally be 60 mg sucrose/mL (6% w/v sucrose), and optimally be ≥80 mgsucrose/mL (8% w/v sucrose), for dissolution following lyophilization.

Polysaccharides formulated in these ways allows conjugation withproteins following lyophilization and redissolution resulting in thedesired properties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates the impact of sodium chloride on conjugate size forS. pneumoniae polysaccharide from serotype 20.

FIG. 2 demonstrates the impact of sodium chloride on lysine consumptionfor S. pneumoniae polysaccharide from serotype 20.

FIG. 3 demonstrates the impact of sodium chloride on free Ps and Pr forS. pneumoniae polysaccharide from serotype 20.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a number of process changes in thepreparation of polysaccharides (Ps) from Streptococcus pneumoniae thatare unique to specific serotypes. These process changes improve theproperties of the polysaccharide and/or the polysaccharide dissolution,resulting in better conjugation.

The inventors have discovered that size reduction of certain S.pneumoniae polysaccharides by acid hydrolysis prior to proteinconjugation yields improved conjugate attributes as shown in theExamples. Without being bound by any particular theory, one possiblemechanism for the use of acid hydrolysis that might explain the observedbehavior is that conjugating with lower molecular mass Ps from acidhydrolysis provides less steric hindrance between Ps and Pr moleculesduring the conjugation reaction, which may in turn lead to enhancedinteraction and improved conjugation.

The inventors have discovered that complete dissolution of certain S.pneumoniae polysaccharides required the presence of sucrose followinglyophilization, as shown in the Examples. Without being bound by anyparticular theory, one possible mechanism for the use of sucrose thatmight explain the observed dissolution behavior is that somepolysaccharide serotypes, due to their chemical structure,self-associate more readily than others during lyophilization ordissolution, and that higher sucrose/polysaccharide ratios inhibitself-association, permitting dissolution following lyophilization.

The inventors have discovered that inclusion of ≥1 mM sodium chlorideprior to or during the conjugation reaction, particularly for S.pneumoniae polysaccharides from serotypes 16F, 20, and 24F, (regardlessof where in the process the sodium chloride is added) results inimproved conjugate attributes such as larger conjugate size, higherlysine consumption, lower free Ps or free Pr, as shown in the Examples.Without being bound by any particular theory, one possible mechanism forthe use of sodium chloride that might explain the observed conjugationbehavior for sodium chloride is that for some polysaccharide serotypes,due to their chemical structure and charge distribution, sodium chlorideprovides electrostatic shielding, allowing enhanced interaction withproteins and leading to improved conjugation. Sodium chloride alsoprovides additional ionic strength to the reaction mixture, which mayreduce the exposure of the hydrophobic region of carrier proteins,thereby reducing protein aggregation.

As used herein, the term “polysaccharide” (Ps) is meant to include anyantigenic saccharide element (or antigenic unit) commonly used in theimmunologic and bacterial vaccine arts, including, but not limited to, a“saccharide”, an “oligosaccharide”, a “polysaccharide”, a“liposaccharide”, a “lipo-oligosaccharide (LOS)”, a “lipopolysaccharide(LPS)”, a “glycosylate”, a “glycoconjugate”, a “derivatized or activatedpolysaccharide or oligosaccharide”, and the like. Unless otherwisespecified, the polysaccharide nomenclature used herein follows theIUB-IUPAC Joint Commission on Biochemical Nomenclature (JCBM)Recommendations 1980. See JCBN, 1982, J. Biol. Chem. 257:3352-3354.

As used herein, “immunogenic composition” refers to a compositioncontaining an antigen, such as a bacterial capsular polysaccharide or apolysaccharide-protein conjugate, that has the ability to elicit animmune response in a host such as a mammal, either humorally orcellularly mediated, or both. The immunogenic composition may serve tosensitize the host by the presentation of the antigen in associationwith MHC molecules at a cell surface. In addition, antigen-specificT-cells or antibodies can be generated to allow for the futureprotection of an immunized host. Immunogenic compositions thus canprotect the host from infection by the bacteria, reduced severity, ormay protect the host from death due to the bacterial infection.Immunogenic compositions may also be used to generate polyclonal ormonoclonal antibodies, which may be used to confer passive immunity to asubject. Immunogenic compositions may also be used to generateantibodies that are functional as measured by the killing of bacteria ineither an animal efficacy model or via an opsonophagocytic killingassay.

As used herein, the term “isolated” in connection with a polysacchariderefers to isolation of S. pneumoniae serotype specific capsularpolysaccharide from purified polysaccharide using purificationtechniques known in the art, including the use of centrifugation, depthfiltration, precipitation, ultrafiltration, treatment with activatecarbon, diafiltration and/or column chromatography. Generally anisolated polysaccharide refers to partial removal of proteins, nucleicacids and non-specific endogenous polysaccharide (C-polysaccharide). Theisolated polysaccharide contains less than 10%, 8%, 6%, 4%, or 2%protein impurities and/or nucleic acids. The isolated polysaccharidecontains less than 20% of C-polysaccharide with respect to type specificpolysaccharides.

As used herein, the term “purified” in connection with a bacterialcapsular polysaccharide refers to the purification of the polysaccharidefrom cell lysate through means such as centrifugation, precipitation,and ultra-filtration. Generally, a purified polysaccharide refers toremoval of cell debris and DNA.

As used herein, the term “Mw” refers to the weight averaged molecularweight and is typically expressed in Da or kDa. Mw takes into accountthat a bigger molecule contains more of the total mass of a polymersample than the smaller molecules do. Mw can be determined by techniquessuch as static light scattering, small angle neutron scattering, X-rayscattering, and sedimentation velocity.

As used herein, the term “Mn” refers to a number average molecularweight and is typically expressed in Da or kDa. Mn is calculated bytaking the total weight of a sample divided by the number of moleculesin the sample and can be determined by techniques such as gel permeationchromatography, viscometry via the (Mark-Houwink equation), colligativemethods such as vapor pressure osmometry, end-group determination orproton NMR. Mw/Mn reflects polydispersity.

As used herein, the term “comprises” when used with the immunogeniccomposition of the invention refers to the inclusion of any othercomponents (subject to limitations of “consisting of” language for theantigen mixture), such as adjuvants and excipients. The term “consistingof” when used with the multivalent polysaccharide-protein conjugatemixture refers to a mixture having those particular S. pneumoniaepolysaccharide protein conjugates and no other S. pneumoniaepolysaccharide protein conjugates from a different serotype.

Unless otherwise specified, all ranges provided herein are inclusive ofthe recited lower and upper limits.

Capsular Polysaccharides

Capsular polysaccharides from Streptococcus pneumoniae from theserotype(s) of the invention (e.g., serotypes 23A, 24F and 31) can beprepared by standard techniques known to those skilled in the art. Forexample, polysaccharides can be isolated from bacteria and may be sizedto some degree by known methods (see, e.g., European Patent Nos.EP497524 and EP497525); and preferably by microfluidisation accomplishedusing a homogenizer or by chemical hydrolysis. In one embodiment, S.pneumoniae strains corresponding to each polysaccharide serotype aregrown in a soy-based medium. The individual polysaccharides are thenpurified through standard steps including centrifugation, precipitation,and ultra-filtration. See, e.g., U.S. Patent Application Publication No.2008/0286838 and U.S. Pat. No. 5,847,112. Polysaccharides can be sizedin order to reduce viscosity and/or to improve filterability ofsubsequent conjugated products. Chemical hydrolysis may be conductedusing acetic acid. Mechanical sizing may be conducted using HighPressure Homogenization Shearing.

For serotypes 12F, 23A, 24F and 31, it was found that homogenization ofpolysaccharide from these serotypes did not result in the desiredcharacteristics. See EXAMPLE 3. Acid hydrolysis can be performed byheating the polysaccharide batch to 80-92° C., preferably 90° C. forserotypes other than 12F, adding an acid such as acetic acid,hydrochloric acid, phosphoric acid, citric acid, to a finalconcentration of 50-200 mM, then incubating for at least 10 minutes, 20minutes, 30 minutes, 40 minutes, or 50 minutes. In certain embodiments,the acid hydrolysis occurs for up to 90 minutes, up to 150 minutes, orup to 155 minutes. At the end of the incubation period, the batch isneutralized by adding, e.g, concentrated potassium phosphate pH 7 bufferto a final concentration of 400 mM and cooling to ≤22° C. Size-reducedpolysaccharide is 0.2-micron filtered and then concentrated anddiafiltered against water using a 5-10 kDa NMWC tangential flowultrafiltration membrane.

In some embodiments, the purified polysaccharides before conjugationhave a molecular weight of between 5 kDa and 4,000 kDa. Molecular weightcan be calculated by size exclusion chromatography (SEC) combined withmultiangle light scattering detector (MALS) and refractive indexdetector (RI). In other such embodiments, the polysaccharide has anaverage molecular weight of between 10 kDa and 4,000 kDa; between 50 kDaand 4,000 kDa; between 50 kDa and 3,000 kDa; between 50 kDa and 2,000kDa; between 50 kDa and 1,500 kDa; between 50 kDa and 1,000 kDa; between50 kDa and 750 kDa; between 50 kDa and 500 kDa; between 100 kDa and4,000 kDa; between 100 kDa and 3,000 kDa; 100 kDa and 2,000 kDa; between100 kDa and 1,500 kDa; between 100 kDa and 1,000 kDa; between 100 kDaand 750 kDa; between 100 kDa and 500 kDa; between 100 and 400 kDa;between 200 kDa and 4,000 kDa; between 200 kDa and 3,000 kDa; between200 kDa and 2,000 kDa; between 200 kDa and 1,500 kDa; between 200 kDaand 1,000 kDa; or between 200 kDa and 500 kDa. In certain embodiments,the average molecular weight is 50-300 kD.

In certain embodiments, the polysaccharide has between 10 and 10,000, 10and 5,000, 10 and 4,000, or 10 and 1000 repeating units. In certainaspects, the polysaccharide has between 20 and 400, 30 to 300, 40 to200, or 50 to 100 repeating units. In certain aspects the polysaccharidehas between 40 and 900 repeating units.

Carrier Protein

Polysaccharides from one or more of the S. pneumoniae serotypesdescribed herein can be conjugated to a carrier protein to improveimmunogenicity in children, the elderly and/or immunocompromisedsubjects. Where more than one serotype is used in a multivalentcomposition, the serotypes may be prepared with the same carrier proteinor different carrier proteins. Each capsular polysaccharide of the sameserotype is typically conjugated to the same carrier protein.

In a particular embodiment of the present invention, CRM197 is used as acarrier protein. CRM197 is a non-toxic variant of diphtheria toxin (DT).The CRM197 carrier protein is a mutant form of DT that is renderednon-toxic by a single amino acid substitution in Fragment A at residue52. In one embodiment, the CRM197 carrier protein is isolated fromcultures of Corynebacterium diphtheria strain C7 (□197) grown incasamino acids and yeast extract-based medium. In another embodiment,CRM197 is prepared recombinantly in accordance with the methodsdescribed in U.S. Pat. No. 5,614,382. Typically, CRM197 is purifiedthrough a combination of ultra-filtration, ammonium sulfateprecipitation, and ion-exchange chromatography. In some embodiments,CRM197 is prepared in Pseudomonas fluorescens using Pfenex ExpressionTechnology™ (Pfenex Inc., San Diego, Calif.).

Other suitable carrier proteins include additional inactivated bacterialtoxins such as DT, Diphtheria toxoid fragment B (DTFB), TT (tetanustoxid) or fragment C of TT, pertussis toxoid, cholera toxoid (e.g., asdescribed in International Patent Application Publication No. WO2004/083251), E. coli LT (heat-labile enterotoxin), E. coli ST(heat-stable enterotoxin), and exotoxin A from Pseudomonas aeruginosa.Bacterial outer membrane proteins such as outer membrane complex c(OMPC), porins, transferrin binding proteins, pneumococcal surfaceprotein A (PspA; See International Application Patent Publication No. WO02/091998), pneumococcal adhesin protein (PsaA), C5a peptidase fromGroup A or Group B streptococcus, or Haemophilus influenzae protein D,pneumococcal pneumolysin (Kuo et al., 1995, Infect Immun 63; 2706-13)including ply detoxified in some fashion for example dPLY-GMBS (SeeInternational Patent Application Publication No. WO 04/081515) ordPLY-formol, PhtX, including PhtA, PhtB, PhtD, PhtE and fusions of Phtproteins for example PhtDE fusions, PhtBE fusions (See InternationalPatent Application Publication Nos. WO 01/98334 and WO 03/54007), canalso be used. Other proteins, such as ovalbumin, keyhole limpethemocyanin (KLH), bovine serum albumin (BSA) or purified proteinderivative of tuberculin (PPD), PorB (from N. meningitidis), PD(Haemophilus influenzae protein D; see, e.g., European Patent No. EP 0594 610 B), or immunologically functional equivalents thereof, syntheticpeptides (See European Patent Nos. EP0378881 and EP0427347), heat shockproteins (See International Patent Application Publication Nos. WO93/17712 and WO 94/03208), pertussis proteins (See International PatentApplication Publication No. WO 98/58668 and European Patent No.EP0471177), cytokines, lymphokines, growth factors or hormones (SeeInternational Patent Application Publication No. WO 91/01146),artificial proteins comprising multiple human CD4+ T cell epitopes fromvarious pathogen derived antigens (See Falugi et al., 2001, Eur JImmunol 31:3816-3824) such as N19 protein (See Baraldoi et al., 2004,Infect Immun 72:4884-7), iron uptake proteins (See International PatentApplication Publication No. WO 01/72337), toxin A or B of C. difficile(See International Patent Publication No. WO 00/61761), and flagellin(See Ben-Yedidia et al., 1998, Immunol Lett 64:9) can also be used ascarrier proteins.

Other DT mutants can also be used as the carrier protein, such asCRM176, CRM228, CRM45 (Uchida et al., 1973, J Biol Chem 218:3838-3844);CRM9, CRM45, CRM102, CRM103 and CRM107 and other mutations described byNicholls and Youle in Genetically Engineered Toxins, Ed: Frankel, MaecelDekker Inc, 1992; deletion or mutation of Glu-148 to Asp, Gln or Serand/or Ala 158 to Gly and other mutations disclosed in U.S. Pat. No.4,709,017 or 4,950,740; mutation of at least one or more residues Lys516, Lys 526, Phe 530 and/or Lys 534 and other mutations disclosed inU.S. Pat. No. 5,917,017 or 6,455,673; or fragment disclosed in U.S. Pat.No. 5,843,711.

Where multivalent vaccines are used, a second carrier protein can beused for one or more of the antigens. The second carrier protein ispreferably a protein that is non-toxic and non-reactogenic andobtainable in sufficient amount and purity. The second carrier proteinis also conjugated or joined with an antigen, e.g., a S. pneumoniaepolysaccharide to enhance immunogenicity of the antigen. Carrierproteins should be amenable to standard conjugation procedures. In oneembodiment, each capsular polysaccharide not conjugated to the firstcarrier protein is conjugated to the same second carrier protein (e.g.,each capsular polysaccharide molecule being conjugated to a singlecarrier protein). In another embodiment, the capsular polysaccharidesnot conjugated to the first carrier protein are conjugated to two ormore carrier proteins (each capsular polysaccharide molecule beingconjugated to a single carrier protein). In such embodiments, eachcapsular polysaccharide of the same serotype is typically conjugated tothe same carrier protein.

Conjugation

Conjugation of pneumococcal polysaccharides to proteins by reductiveamination in an aprotic solvent such as DMSO is commonly used. Activatedpolysaccharides (Ps) and proteins (Pr) are typically lyophilized,resuspended in DMSO, then combined with sodium cyanoborohydride andsodium borohydride added to achieve conjugation. Process details areprovided below.

For many pneumococcal serotypes, this process yields conjugates thatmeet target attributes for size, lysine consumption, freepolysaccharide, and free protein. However for some serotypes it wasfound that target conjugate attributes were more difficult to achievewith this DMSO process, even after optimizing conjugation parameterssuch as Ps and Pr concentrations and conjugation time. See the EXAMPLES.As described below, the present invention provides several solutions toovercome these issues.

Prior to conjugation, the purified polysaccharides can be chemicallyactivated to make the saccharides capable of reacting with the carrierprotein to form an activated polysaccharide. As used herein, the term“activated polysaccharide” refers to a polysaccharide that has beenchemically modified as described below to enable conjugation to a linkeror a carrier protein. The purified polysaccharides can optionally beconnected to a linker. Once activated or connected to a linker, eachcapsular polysaccharide is separately conjugated to a carrier protein toform a glycoconjugate. The polysaccharide conjugates may be prepared byknown coupling techniques.

In certain embodiments, the polysaccharide can be coupled to a linker toform a polysaccharide-linker intermediate in which the free terminus ofthe linker is an ester group. The linker is therefore one in which atleast one terminus is an ester group. The other terminus is selected sothat it can react with the polysaccharide to form thepolysaccharide-linker intermediate.

In certain embodiments, the polysaccharide can be coupled to a linkerusing a primary amine group in the polysaccharide. In this case, thelinker typically has an ester group at both termini. This allows thecoupling to take place by reacting one of the ester groups with theprimary amine group in the polysaccharide by nucleophilic acylsubstitution. The reaction results in a polysaccharide-linkerintermediate in which the polysaccharide is coupled to the linker via anamide linkage. The linker is therefore a bifunctional linker thatprovides a first ester group for reacting with the primary amine groupin the polysaccharide and a second ester group for reacting with theprimary amine group in the carrier molecule. A typical linker is adipicacid N-hydroxysuccinimide diester (SIDEA).

In certain embodiments, the coupling can also take place indirectly,i.e. with an additional linker that is used to derivatise thepolysaccharide prior to coupling to the linker. The polysaccharide iscoupled to the additional linker using a carbonyl group at the reducingterminus of the polysaccharide. This coupling comprises two steps: (a1)reacting the carbonyl group with the additional linker; and (a2)reacting the free terminus of the additional linker with the linker. Inthese embodiments, the additional linker typically has a primary aminegroup at both termini, thereby allowing step (a1) to take place byreacting one of the primary amine groups with the carbonyl group in thepolysaccharide by reductive amination. A primary amine group is usedthat is reactive with the carbonyl group in the polysaccharide.Hydrazide or hydroxylamino groups are suitable. The same primary aminegroup is typically present at both termini of the additional linker. Thereaction results in a polysaccharide-additional linker intermediate inwhich the polysaccharide is coupled to the additional linker via a C—Nlinkage.

In certain embodiments, the polysaccharide can be coupled to theadditional linker using a different group in the polysaccharide,particularly a carboxyl group. This coupling comprises two steps: (a1)reacting the group with the additional linker; and (a2) reacting thefree terminus of the additional linker with the linker. In this case,the additional linker typically has a primary amine group at bothtermini, thereby allowing step (a1) to take place by reacting one of theprimary amine groups with the carboxyl group in the polysaccharide byEDAC activation. A primary amine group is used that is reactive with theEDAC-activated carboxyl group in the polysaccharide. A hydrazide groupis suitable. The same primary amine group is typically present at bothtermini of the additional linker. The reaction results in apolysaccharide-additional linker intermediate in which thepolysaccharide is coupled to the additional linker via an amide linkage.

In one embodiment, the chemical activation of the polysaccharides andsubsequent conjugation to the carrier protein by reductive amination canbe achieved by means described in U.S. Pat. Nos. 4,365,170, 4,673,574and 4,902,506, U.S. Patent Application Publication Nos. 2006/0228380,2007/184072, 2007/0231340 and 2007/0184071, and International PatentApplication Publication Nos. WO2006/110381, WO2008/079653, andWO2008/143709). The chemistry may entail the activation of pneumococcalpolysaccharide by reaction with any oxidizing agent which a primaryhydroxyl group to an aldehyde, such as TEMPO in the presence of oxidant(WO2104/097099), or reacting two vicinal hydroxyl groups to aldehydes,such as periodate (including sodium periodate, potassium periodate, orperiodic acid). The reactions lead to a random oxidation of primaryhydroxyl groups or random oxidative cleavage of vicinal hydroxyl groupsof the carbohydrates with the formation of reactive aldehyde groups.

In this embodiment, coupling to the carrier protein is by reductiveamination via direct amination to the lysyl groups of the protein. Forexample, conjugation is carried out by reacting a mixture of theactivated polysaccharide and carrier protein with a reducing agent suchas sodium cyanoborohydride, optionally in the presence of nickel foraqueous conjugation. The conjugation reaction may take place underaqueous solution or in the presence of dimethylsulfoxide (DMSO). See,e.g., U.S. Patent Application Publication Nos. US2015/0231270 andUS2011/0195086 and European Patent No. EP 0471 177 B1. Unreactedaldehydes are then capped with the addition of a strong reducing agent,such as sodium borohydride.

Reductive amination involves two steps, (1) oxidation of thepolysaccharide to form reactive aldehydes, (2) reduction of the imine(Schiff base) formed between activated polysaccharide and a carrierprotein to form a stable amine conjugate bond. Before oxidation, thepolysaccharide is optionally size reduced. Mechanical methods (e.g.homogenization) or chemical hydrolysis may be employed. Chemicalhydrolysis may be conducted using acetic acid. The oxidation step mayinvolve reaction with periodate. For the purpose of the presentinvention, the term “periodate” includes both periodate and periodicacid; the term also includes both metaperiodate (IO₄ ⁻) andorthoperiodate (IO₆ ⁵⁻) and includes the various salts of periodate(e.g., sodium periodate and potassium periodate). In an embodiment thecapsular polysaccharide is oxidized in the presence of metaperiodate,preferably in the presence of sodium periodate (NaIO₄). In anotherembodiment the capsular polysaccharide is oxydized in the presence oforthoperiodate, preferably in the presence of periodic acid.

In an embodiment, the oxidizing agent is a stable nitroxyl or nitroxideradical compound, such as piperidine-N-oxy or pyrrolidine-N-oxycompounds, in the presence of an oxidant to selectively oxidize primaryhydroxyls (as described in, for example, International PatentApplication Publication No. WO 2014/097099). In said reaction, theactual oxidant is the N-oxoammonium salt, in a catalytic cycle. In anaspect, said stable nitroxyl or nitroxide radical compound arepiperidine-N-oxy or pyrrolidine-N-oxy compounds. In an aspect, saidstable nitroxyl or nitroxide radical compound bears a TEMPO(2,2,6,6-tetramethyl-1-piperidinyloxy) or a PROXYL(2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety. In an aspect, saidstable nitroxyl radical compound is TEMPO or a derivative thereof. In anaspect, said oxidant is a molecule bearing a N-halo moiety. In anaspect, said oxidant is selected from the group consisting ofN-ChloroSuccinimide, N-Bromosuccinimide, N-Iodosuccinimide,Dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione,Dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinane-2,4,6-trione,Diiodoisocyanuric acid and 1,3,5-triiodo-1,3,5-triazinane-2,4,6-trione.Preferably said oxidant is N-Chlorosuccinimide.

In certain aspects, the oxidizing agent is2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO) free radical andN-Chlorosuccinimide (NCS) as the cooxidant (as described inInternational Patent Application Publication No. WO2014/097099).Therefore in one aspect, the glycoconjugates from S. pneumoniae areobtainable by a method comprising the steps of: a) reacting a saccharidewith 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) andN-chlorosuccinimide (NCS) in an aqueous solvent to produce an activatedsaccharide; and b) reacting the activated saccharide with a carrierprotein comprising one or more amine groups (said method is designated“TEMPO/NCS-reductive amination” thereafter).

Optionally the oxidation reaction is quenched by addition of a quenchingagent. The quenching agent maybe selected from vicinal diols,1,2-aminoalcohols, amino acids, glutathione, sulfite, bisulfate,dithionite, metabisulfite, thiosulfate, phosphites, hypophosphites orphosphorous acid (such as glycerol, ethylene glycol, propan-1,2-diol,butan-1,2-diol or butan-2,3-diol, ascorbic acid).

The second step of the conjugation process for reductive amination isthe reduction of the imine (Schiff base) bond between activatedpolysaccharide and a carrier protein to form a stable conjugate bond(so-called reductive amination), using a reducing agent. Reducing agentswhich are suitable include the cyanoborohydrides (such as sodiumcyanoborohydride) or sodium borohydride. In one embodiment the reducingagent is sodium cyanoborohydride.

In certain embodiments of the methods of the invention, the reductiveamination reaction is carried out in aprotic solvent (or a mixture ofaprotic solvents). In an embodiment, the reduction reaction is carriedout in DMSO (dimethylsulfoxide) or in DMF (dimethylformamide) solvent.The DMSO or DMF solvent may be used to reconstitute the activatedpolysaccharide and carrier protein, if lyophilized. In one embodiment,the aprotic solvent is DMSO.

Sucrose at up to 5%, at a sucrose:Ps mass ratio of 25×, has been used toachieve optimal dissolution in DMSO following lyophilization. See, e.g.,International Patent Application Publication No. WO2017/013548. For S.pneumoniae polysaccharides obtained from serotypes 3, 8 and 24F, it wasfound that higher levels of sucrose were needed to adequately dissolvethe polysaccharide prior to protein conjugation in an aprotic solvent.In some embodiments, for these serotypes, sucrose concentrations greaterthan 5% in an aqueous solution are used. In some embodiments, for theseserotypes, sucrose:Ps mass ratios greater than 25× are used, e.g., atleast 30×, at least 35×, or at least 40×. In some embodiments, thepre-lyophilization mass ratio of sucrose to polysaccharide is greaterthan or equal to 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50 ormore. Other sugars such as trehalose or mannitol can be used.

It was also found that the presence of sodium chloride (NaCl) in theprotein conjugation process, whether in an aqueous solution or anaprotic solvent, can be used to reduce the free protein levels, increaseconjugate molecular weight, lower free polysaccharide and/or increaselysine consumption for S. pneumoniae polysaccharides purified fromserotypes 15A, 16F, 17F, 20, 23A, 24F, and 35B. Thus, the presentinvention can be directed to a method for preparing a polysaccharideprotein conjugate, the method comprising reacting a S. pneumoniaepolysaccharide within a first solution with a protein within a secondsolution to form a third solution in which the polysaccharide proteinconjugate reaction takes place to form the polysaccharide proteinconjugate, wherein the third solution comprises at least 1 mM salt.

The sodium chloride can be added anywhere in the conjugation processfrom the preparation of polysaccharide and protein for lyophilizationprior to the conjugation reaction to the conjugation reaction itself,e.g., during the Schiff base reaction or during the reductive aminationin the presence of sodium cyanoborohydride. In some embodiments, thepolysaccharide and protein are separately lyophilized and the salt canbe added to either the polysaccharide solution (first solution) or theprotein solution (second solution) or both. In some embodiments, thepolysaccharide and protein are lyophilized from the same solution towhich a salt is added (i.e., the first and second solution are thesame). In some embodiments, the salt is added into the solution in whichthe polysaccharide protein conjugate reaction takes place (i.e., thethird solution)

Other salts can be used such as other sodium salts, potassium salts suchas potassium chloride, lithium salts, magnesium salts and calcium salts.In some embodiments, from 1 mM to 100 mM of sodium chloride is added tothe dissolution solution for the polysaccharide or during the Schiffbase reaction, or during the reductive amination reaction in thepresence of sodium cyanoborohydride. In some embodiments, at least 2, 3,4, 5, 6, 7, 8, 9 or 10 mM sodium chloride is used. In some aspects ofthese embodiments, no more than 100, 75, or 50 mM sodium chloride isused.

At the end of the reduction reaction, there may be unreacted aldehydegroups remaining in the conjugates, which may be capped or quenchedusing a suitable capping or quenching agent. In one embodiment thiscapping or quenching agent is sodium borohydride (NaBH₄). Suitablealternatives include sodium triacetoxyborohydride or sodium or zincborohydride in the presence of Bronsted or Lewis acids, amine boranessuch as pyridine borane, 2-Picoline Borane, 2,6-diborane-methanol,dimethylamine-borane, t-BuMe′PrN-BH₃, benzylamine-BH₃ or5-ethyl-2-methylpyridine borane (PEMB) or borohydride exchange resin.

Glycoconjugates prepared using reductive amination in an aprotic solventare generally used in multivalent pneumococcal protein conjugatevaccines. Thus, in certain embodiments for multivalent compositionswhere not all the serotypes are prepared in an aprotic solvent, thereduction reaction for the remaining serotypes is carried out in aqueoussolvent (e.g., selected from PBS (phosphate buffered saline), MES(2-(N-morpholino)ethanesulfonic acid), HEPES,(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Bis-tris, ADA(N-(2-Acetamido)iminodiacetic acid), PIPES(piperazine-N,N′-bis(2-ethanesulfonic acid)), MOPSO(3-Morpholino-2-hydroxypropanesulfonic acid), BES(N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), MOPS(3-(N-morpholino)propanesulfonic acid), DIPSO (3-Bis(2-hydroxyethyl)amino-2-hydroxypropane-1-sulfonic acid), MOBS(4-(N-morpholino)butanesulfonic acid), HEPPSO(N-(2-Hydroxyethyl)piperazine-N-(2-hydroxypropanesulfonic acid)), POPSO(Piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid)), TEA(triethanolamine), EPPS (4-(2-Hydroxyethyl)piperazine-1-propanesulfonicacid), Bicine or HEPB, at a pH between 6.0 and 8.5, 7.0 and 8.0, or 7.0and 7.5).

In some embodiments, the glycoconjugates of the present inventioncomprise a polysaccharide having a molecular weight of between 10 kDaand 10,000 kDa. In other such embodiments, the polysaccharide has amolecular weight of between 25 kDa and 5,000 kDa. In other suchembodiments, the polysaccharide has a molecular weight of between 50 kDaand 1,000 kDa. In other such embodiments, the polysaccharide has amolecular weight of between 70 kDa and 900 kDa. In other suchembodiments, the polysaccharide has a molecular weight of between 100kDa and 800 kDa. In other such embodiments, the polysaccharide has amolecular weight of between 200 kDa and 600 kDa. In further suchembodiments, the polysaccharide has a molecular weight of 100 kDa to1000 kDa; 100 kDa to 900 kDa; 100 kDa to 800 kDa; 100 kDa to 700 kDa;100 kDa to 600 kDa; 100 kDa to 500 kDa; 100 kDa to 400 kDa; 100 kDa to300 kDa; 150 kDa to 1,000 kDa; 150 kDa to 900 kDa; 150 kDa to 800 kDa;150 kDa to 700 kDa; 150 kDa to 600 kDa; 150 kDa to 500 kDa; 150 kDa to400 kDa; 150 kDa to 300 kDa; 200 kDa to 1,000 kDa; 200 kDa to 900 kDa;200 kDa to 800 kDa; 200 kDa to 700 kDa; 200 kDa to 600 kDa; 200 kDa to500 kDa; 200 kDa to 400 kDa; 200 kDa to 300; 250 kDa to 1,000 kDa; 250kDa to 900 kDa; 250 kDa to 800 kDa; 250 kDa to 700 kDa; 250 kDa to 600kDa; 250 kDa to 500 kDa; 250 kDa to 400 kDa; 250 kDa to 350 kDa; 300 kDato 1,000 kDa; 300 kDa to 900 kDa; 300 kDa to 800 kDa; 300 kDa to 700kDa; 300 kDa to 600 kDa; 300 kDa to 500 kDa; 300 kDa to 400 kDa; 400 kDato 1,000 kDa; 400 kDa to 900 kDa; 400 kDa to 800 kDa; 400 kDa to 700kDa; 400 kDa to 600 kDa; or 500 kDa to 600 kDa. In certain embodimentswhere acid hydrolysis is employed, the polysaccharide has a molecularweight of between 10 kDa and 200 kDa, 25 kDa and 200 kDa, 50 kDa and 200kDa, 10 kDa and 150 kDa, 25 kDa and 150 kDa, or 50 kDa and 150 kDa.

Suitable alternative chemistries include the activation of thesaccharide with 1-cyano-4-dimethylamino pyridinium tetrafluoroborate(CDAP) to form a cyanate ester. The activated saccharide may thus becoupled directly or via a spacer (linker) group to an amino group on thecarrier protein. For example, the spacer could be cystamine orcysteamine to give a thiolated polysaccharide which could be coupled tothe carrier via a thioether linkage obtained after reaction with amaleimide-activated carrier protein (for example using GMBS) or ahaloacetylated carrier protein (for example using iodoacetimide [e.g.ethyl iodoacetimide HCl] or N-succinimidyl bromoacetate or SIAB, or SIA,or SBAP). Preferably, the cyanate ester (optionally made by CDAPchemistry) is coupled with hexane diamine or adipic acid dihydrazide(ADH) and the amino-derivatised saccharide is conjugated to the carrierprotein using carbodiimide (e.g. EDAC or EDC) chemistry via a carboxylgroup on the protein carrier. Such conjugates are described inInternational Patent Application Publication Nos. WO 93/15760, WO95/08348 and WO 96/29094; and Chu et al., 1983, Infect. Immunity40:245-256.

Other suitable techniques use carbodiimides, hydrazides, active esters,norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S—NHS, EDC, TSTU.Many are described in International Patent Application Publication No.WO 98/42721. Conjugation may involve a carbonyl linker which may beformed by reaction of a free hydroxyl group of the saccharide with CDI(See Bethell et al., 1979, J. Biol. Chem. 254:2572-4; Hearn et al.,1981, J. Chromatogr. 218:509-18) followed by reaction with a protein toform a carbamate linkage. This may involve reduction of the anomericterminus to a primary hydroxyl group, optional protection/deprotectionof the primary hydroxyl group, reaction of the primary hydroxyl groupwith CDI to form a CDI carbamate intermediate and coupling the CDIcarbamate intermediate with an amino group on a protein.

Following the conjugation (the reduction reaction and optionally thecapping or quenching reaction), the glycoconjugates may be purified(enriched with respect to the amount of polysaccharide-proteinconjugate) by a variety of techniques known to the skilled person. Thesetechniques include dialysis, concentration/diafiltration operations,tangential flow filtration, ultrafiltration, precipitation/elution,column chromatography (ion exchange chromatography, multimodal ionexchange chromatography, DEAE, or hydrophobic interactionchromatography), and depth filtration. See, e.g., U.S. Pat. No.6,146,902. In an embodiment, the glycoconjugates are purified bydiafilitration or ion exchange chromatography or size exclusionchromatography.

One way to characterize the glycoconjugates of the invention is by thenumber of lysine residues in the carrier protein (e.g., CRM197) thatbecome conjugated to the saccharide, which can be characterized as arange of conjugated lysines (degree of conjugation). The evidence forlysine modification of the carrier protein, due to covalent linkages tothe polysaccharides, can be obtained by amino acid analysis usingroutine methods known to those of skill in the art. Conjugation resultsin a reduction in the number of lysine residues recovered, compared tothe carrier protein starting material used to generate the conjugatematerials. In a preferred embodiment, the degree of conjugation of theglycoconjugate of the invention is between 2 and 15, between 2 and 13,between 2 and 10, between 2 and 8, between 2 and 6, between 2 and 5,between 2 and 4, between 3 and 15, between 3 and 13, between 3 and 10,between 3 and 8, between 3 and 6, between 3 and 5, between 3 and 4,between 5 and 15, between 5 and 10, between 8 and 15, between 8 and 12,between 10 and 15 or between 10 and 12. In an embodiment, the degree ofconjugation of the glycoconjugate of the invention is about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, about 10, about11, about 12, about 13, about 14 or about 15. In a preferred embodiment,the degree of conjugation of the glycoconjugate of the invention isbetween 7 and 12. In some such embodiments, the carrier protein isCRM197.

The glycoconjugates of the invention may also be characterized by theratio (weight/weight) of saccharide to carrier protein. In someembodiments, the ratio of polysaccharide to carrier protein in theglycoconjugate (w/w) is between 0.5 and 3.0 (e.g., about 0.5, about 0.6,about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9,about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about2.6, about 2.7, about 2.8, about 2.9, or about 3.0). In otherembodiments, the saccharide to carrier protein ratio (w/w) is between0.5 and 2.0, between 0.5 and 1.5, between 0.8 and 1.2, between 0.5 and1.0, between 1.0 and 1.5 or between 1.0 and 2.0. In further embodiments,the saccharide to carrier protein ratio (w/w) is between 0.8 and 1.2. Ina preferred embodiment, the ratio of capsular polysaccharide to carrierprotein in the conjugate is between 1 and 2. In some such embodiments,the carrier protein is CRM197. The glycoconjugates and immunogeniccompositions of the invention may contain free saccharide that is notcovalently conjugated to the carrier protein, but is neverthelesspresent in the glycoconjugate composition. The free saccharide may benon-covalently associated with (i.e., non-covalently bound to, adsorbedto, or entrapped in or with) the glycoconjugate.

In a preferred embodiment, the glycoconjugate comprises less than about50%, 45%, 40%, 35%, 30%, 25%, 20% or 15% of free polysaccharide comparedto the total amount of polysaccharide. In a preferred embodiment theglycoconjugate comprises less than about 25% of free polysaccharidecompared to the total amount of polysaccharide. In a preferredembodiment the glycoconjugate comprises less than about 20% of freepolysaccharide compared to the total amount of polysaccharide. In apreferred embodiment the glycoconjugate comprises less than about 15% offree polysaccharide compared to the total amount of polysaccharide.

Multivalent Polysaccharide-Protein Conjugate Vaccines

Polysaccharide-protein conjugates prepared using the methods of theinvention can be used in multivalent polysaccharide-protein conjugatevaccines. In certain embodiments, multivalent polysaccharide-proteinconjugate vaccines comprise S. pneumoniae capsular polysaccharides fromone or more of S. pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D,7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18B,18C, 19A, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 28A, 31,33F, 34, 35A, 35B, 35F, and 38 either as free polysaccharides, acomponent of a polysaccharide-protein conjugate or a combinationthereof, to provide a multivalent pneumococcal vaccine. In certainembodiments, the immunogenic composition comprises, consists essentiallyof, or consists of S. pneumoniae capsular polysaccharides from 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, or 44 S. pneumoniae serotypes individually conjugated to one ormore carrier proteins. Preferably, saccharides from a particularserotype are not conjugated to more than one carrier protein.

After the individual glycoconjugates are purified, they are compoundedto formulate the immunogenic composition of the present invention. Thesepneumococcal conjugates are prepared by separate processes and bulkformulated into a single dosage formulation.

Pharmaceutical/Vaccine Compositions

The present invention further provides compositions, includingpharmaceutical, immunogenic and vaccine compositions, comprising,consisting essentially of, or alternatively, consisting of any of thepolysaccharide S. pneumoniae serotype combinations described abovetogether with a pharmaceutically acceptable carrier and an adjuvant.

Formulation of the polysaccharide-protein conjugates can be accomplishedusing art-recognized methods. For instance, individual pneumococcalconjugates can be formulated with a physiologically acceptable vehicleto prepare the composition. Examples of such vehicles include, but arenot limited to, water, buffered saline, polyols (e.g., glycerol,propylene glycol, liquid polyethylene glycol) and dextrose solutions.

In a preferred formulation, the vaccine composition is formulated inL-histidine buffer with sodium chloride.

As defined herein, an “adjuvant” is a substance that serves to enhancethe immunogenicity of an immunogenic composition of the invention. Animmune adjuvant may enhance an immune response to an antigen that isweakly immunogenic when administered alone, e.g., inducing no or weakantibody titers or cell-mediated immune response, increase antibodytiters to the antigen, and/or lowers the dose of the antigen effectiveto achieve an immune response in the individual. Thus, adjuvants areoften given to boost the immune response and are well known to theskilled artisan. Suitable adjuvants to enhance effectiveness of thecomposition include, but are not limited to:

(1) aluminum salts (alum), such as aluminum hydroxide, aluminumphosphate, aluminum sulfate, etc.;

(2) oil-in-water emulsion formulations (with or without other specificimmunostimulating agents such as muramyl peptides (defined below) orbacterial cell wall components), such as, for example, (a) MF59(International Patent Application Publication No. WO 90/14837),containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionallycontaining various amounts of MTP-PE) formulated into submicronparticles using a microfluidizer such as Model 110Y microfluidizer(Microfluidics, Newton, Mass.), (b) SAF, containing 10% Squalene, 0.4%Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion, (c) Ribi™ adjuvant system (RAS), (Corixa,Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or morebacterial cell wall components from the group consisting of3-O-deacylated monophosphorylipid A (MPL™) described in U.S. Pat. No.4,912,094, trehalose dimycolate (TDM), and cell wall skeleton (CWS),preferably MPL+CWS (Detox™); and (d) a Montanide ISA;

(3) saponin adjuvants, such as Quil A or STIMULON™ QS-21 (Antigenics,Framingham, Mass.) (see, e.g., U.S. Pat. No. 5,057,540) may be used orparticles generated therefrom such as ISCOM (immunostimulating complexesformed by the combination of cholesterol, saponin, phospholipid, andamphipathic proteins) and Iscomatrix® (having essentially the samestructure as an ISCOM but without the protein);

(4) bacterial lipopolysaccharides, synthetic lipid A analogs such asaminoalkyl glucosamine phosphate compounds (AGP), or derivatives oranalogs thereof, which are available from Corixa, and which aredescribed in U.S. Pat. No. 6,113,918; one such AGP is2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl2-Deoxy-4-O-phosphono-3-O—[(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-b-D-glucopyranoside,which is also known as 529 (formerly known as RC529), which isformulated as an aqueous form or as a stable emulsion

(5) synthetic polynucleotides such as oligonucleotides containing CpGmotif(s) (U.S. Pat. No. 6,207,646);

(6) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6,IL-7, IL-12, IL-15, IL-18, etc.), interferons (e.g., gamma interferon),granulocyte macrophage colony stimulating factor (GM-CSF), macrophagecolony stimulating factor (M-CSF), tumor necrosis factor (TNF),costimulatory molecules B7-1 and B7-2, etc; and

(7) complement, such as a trimer of complement component C3d.

In another embodiment, the adjuvant is a mixture of 2, 3, or more of theabove adjuvants, e.g., SBAS2 (an oil-in-water emulsion also containing3-deacylated monophosphoryl lipid A and QS21).

Muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanine-2-(1′,2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

In certain embodiments, the adjuvant is an aluminum salt. The aluminumsalt adjuvant may be an alum-precipitated vaccine or an alum-adsorbedvaccine. Aluminum-salt adjuvants are well known in the art and aredescribed, for example, in Harlow, E. and D. Lane (1988; Antibodies: ALaboratory Manual Cold Spring Harbor Laboratory) and Nicklas, W. (1992;Aluminum salts. Research in Immunology 143:489-493). The aluminum saltincludes, but is not limited to, hydrated alumina, alumina hydrate,alumina trihydrate (ATH), aluminum hydrate, aluminum trihydrate,Alhydrogel®, Superfos, Amphogel®, aluminum (III) hydroxide, aluminumhydroxyphosphate (Aluminum Phosphate Adjuvant (APA)), amorphous alumina,trihydrated alumina, or trihydroxyaluminum.

APA is an aqueous suspension of aluminum hydroxyphosphate. APA ismanufactured by blending aluminum chloride and sodium phosphate in a 1:1volumetric ratio to precipitate aluminum hydroxyphosphate. After theblending process, the material is size-reduced with a high-shear mixerto achieve a monodisperse particle size distribution. The product isthen diafiltered against physiological saline and steam sterilized.

In certain embodiments, a commercially available Al(OH)₃ (e.g.Alhydrogel® or Superfos of Denmark/Accurate Chemical and Scientific Co.,Westbury, N.Y.) is used to adsorb proteins. Adsorption of protein isdependent, in another embodiment, on the pI (Isoelectric pH) of theprotein and the pH of the medium. A protein with a lower pI adsorbs tothe positively charged aluminum ion more strongly than a protein with ahigher pI. Aluminum salts may establish a depot of Ag that is releasedslowly over a period of 2-3 weeks, be involved in nonspecific activationof macrophages and complement activation, and/or stimulate innate immunemechanism (possibly through stimulation of uric acid). See, e.g.,Lambrecht et al., 2009, Curr Opin Immunol 21:23.

Monovalent bulk aqueous conjugates are typically blended together anddiluted. Once diluted, the batch is sterile filtered. Aluminum phosphateadjuvant is added aseptically to target a final concentration of 4 μg/mLfor all S. pneumoniae serotypes except serotype 6B, which is diluted toa target of 8 μg/mL, and a final aluminum concentration of 250 μg/mL.The adjuvanted, formulated batch will be filled into vials or syringes.

In certain embodiments, the adjuvant is a CpG-containing nucleotidesequence, for example, a CpG-containing oligonucleotide, in particular,a CpG-containing oligodeoxynucleotide (CpG ODN). In another embodiment,the adjuvant is ODN 1826, which may be acquired from ColeyPharmaceutical Group.

“CpG-containing nucleotide,” “CpG-containing oligonucleotide,” “CpGoligonucleotide,” and similar terms refer to a nucleotide molecule of6-50 nucleotides in length that contains an unmethylated CpG moiety.See, e.g., Wang et al., 2003, Vaccine 21:4297. In another embodiment,any other art-accepted definition of the terms is intended.CpG-containing oligonucleotides include modified oligonucleotides usingany synthetic internucleoside linkages, modified base and/or modifiedsugar.

Methods for use of CpG oligonucleotides are well known in the art andare described, for example, in Sur et al., 1999, J Immunol. 162:6284-93;Verthelyi, 2006, Methods Mol Med. 127:139-58; and Yasuda et al., 2006,Crit Rev Ther Drug Carrier Syst. 23:89-110.

Administration/Dosage

The compositions and formulations described herein can be used toprotect or treat a human susceptible to infection, e.g., a pneumococcalinfection, by means of administering the vaccine via a systemic ormucosal route. For example, the compositions and formulations describedherein can be used in a method of inducing an immune response to a S.pneumoniae capsular polysaccharide conjugate, comprising administeringto a human an immunologically effective amount of an immunogeniccomposition or formulation described herein. In another example, thecompositions and formulations described herein can be used in a methodof vaccinating a human against a pneumococcal infection, comprising thestep of administering to the human an immunogically effective amount ofan immunogenic composition or formulation described herein.

Optimal amounts of components for a particular vaccine can beascertained by standard studies involving observation of appropriateimmune responses in subjects. For example, in another embodiment, thedosage for human vaccination is determined by extrapolation from animalstudies to human data. In another embodiment, the dosage is determinedempirically.

“Effective amount” of a composition of the invention refers to a doserequired to elicit antibodies that significantly reduce the likelihoodor severity of infectivity of a microbe, e.g., S. pneumoniae, during asubsequent challenge.

Methods using the compositions and formulations described herein can beused for the prevention and/or reduction of primary clinical syndromescaused by microbes, e.g., S. pneumoniae, including both invasiveinfections (meningitis, pneumonia, and bacteremia), and noninvasiveinfections (acute otitis media, and sinusitis).

Administration of the compositions and formulation described herein caninclude one or more of: injection via the intramuscular,intraperitoneal, intradermal or subcutaneous routes; or via mucosaladministration to the oral/alimentary, respiratory or genitourinarytracts. In one embodiment, intranasal administration is used for thetreatment of pneumonia or otitis media (as nasopharyngeal carriage ofpneumococci can be more effectively prevented, thus attenuatinginfection at its earliest stage).

The amount of conjugate in each vaccine dose is selected as an amountthat induces an immunoprotective response without significant, adverseeffects. Such amount can vary depending upon the pneumococcal serotype.Generally, for polysaccharide-based conjugates, each dose will comprise0.1 to 100 □g of each polysaccharide, particularly 0.1 to 10 □g, andmore particularly 1 to 5 □g. For example, each dose can comprise 100,150, 200, 250, 300, 400, 500, or 750 ng or 1, 1.5, 2, 3, 4, 5, 6, 7,7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 25, 30, 40, 50, 60,70, 80, 90, or 100 □g of each polysaccharide.

Optimal amounts of components for a particular vaccine can beascertained by standard studies involving observation of appropriateimmune responses in subjects. For example, in another embodiment, thedosage for human vaccination is determined by extrapolation from animalstudies to human data. In another embodiment, the dosage is determinedempirically.

In one embodiment, the dose of the aluminum salt is 10, 15, 20, 25, 30,50, 70, 100, 125, 150, 200, 300, 500, or 700 □g, or 1, 1.2, 1.5, 2, 3, 5mg or more. In yet another embodiment, the dose of aluminum saltdescribed above is per □g of recombinant protein.

Generally, each 0.5 mL dose is formulated to contain: 2 □g of each S.pneumoniae polysaccharide, except for serotype 6B polysaccharide at 4□g; about 32 □g CRM197 carrier protein (e.g., 32 □g±5 □g, ±3 □□g, ±2 □g,or ±1 □g); 0.125 mg of elemental aluminum (0.5 mg aluminum phosphate)adjuvant; and sodium chloride and L-histidine buffer. The sodiumchloride concentration is about 150 mM (e.g., 150 mM±25 mM, ±20 mM, ±15mM, ±10 mM, or ±5 mM) and about 20 mM (e.g., 20 mM±5 mM, ±2.5 mM, ±2 mM,±1 mM, or ±0.5 mM) L-histidine buffer.

According to any of the methods using a composition or formulationdescribed herein, and in one embodiment, the subject is human. Incertain embodiments, the human patient is an infant (less than 1 year ofage), toddler (approximately 12 to 24 months), or young child(approximately 2 to 5 years). In other embodiments, the human patient isan elderly patient (>65 years). The compositions of this invention arealso suitable for use with older children, adolescents and adults (e.g.,aged 18 to 45 years or 18 to 65 years).

In one embodiment of the methods using a composition or formulationdescribed herein, a composition or formulation is administered as asingle inoculation. In another embodiment, the composition orformulation is administered twice, three times or four times or more,adequately spaced apart. For example, the composition or formulation maybe administered at 1, 2, 3, 4, 5, or 6 month intervals or anycombination thereof. The immunization schedule can follow thatdesignated for pneumococcal vaccines. For example, the routine schedulefor infants and toddlers against invasive disease caused by S.pneumoniae is 2, 4, 6 and 12-15 months of age. Thus, in a preferredembodiment, the composition is administered as a 4-dose series at 2, 4,6, and 12-15 months of age.

The compositions described herein may also include one or more proteinsfrom S. pneumoniae. Examples of S. pneumoniae proteins suitable forinclusion include those identified in International Patent ApplicationPublication Nos. WO 02/083855 and WO 02/053761.

Formulations

The compositions described herein can be administered to a subject byone or more method known to a person skilled in the art, such asparenterally, transmucosally, transdermally, intramuscularly,intravenously, intra-dermally, intra-nasally, subcutaneously,intra-peritonealy, and formulated accordingly.

In one embodiment, compositions described herein are administered viaepidermal injection, intramuscular injection, intravenous,intra-arterial, subcutaneous injection, or intra-respiratory mucosalinjection of a liquid preparation. Liquid formulations for injectioninclude solutions and the like.

The composition can be formulated as single dose vials, multi-dose vialsor as pre-filled syringes.

In another embodiment, compositions are administered orally, and arethus formulated in a form suitable for oral administration, i.e., as asolid or a liquid preparation. Solid oral formulations include tablets,capsules, pills, granules, pellets and the like. Liquid oralformulations include solutions, suspensions, dispersions, emulsions,oils and the like.

Pharmaceutically acceptable carriers for liquid formulations are aqueousor nonaqueous solutions, suspensions, emulsions or oils. Examples ofnonaqueous solvents are propylene glycol, polyethylene glycol, andinjectable organic esters such as ethyl oleate. Aqueous carriers includewater, alcoholic/aqueous solutions, emulsions or suspensions, includingsaline and buffered media. Examples of oils are those of animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil,olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipidfrom milk or eggs.

The pharmaceutical composition may be isotonic, hypotonic or hypertonic.However, it is often preferred that a pharmaceutical composition forinfusion or injection is essentially isotonic when it is administrated.Hence, for storage the pharmaceutical composition may preferably beisotonic or hypertonic. If the pharmaceutical composition is hypertonicfor storage, it may be diluted to become an isotonic solution prior toadministration.

The isotonic agent may be an ionic isotonic agent such as a salt or anon-ionic isotonic agent such as a carbohydrate. Examples of ionicisotonic agents include but are not limited to NaCl, CaCl₂, KCl andMgCl₂. Examples of non-ionic isotonic agents include but are not limitedto sucrose, trehalose, mannitol, sorbitol and glycerol.

It is also preferred that at least one pharmaceutically acceptableadditive is a buffer. For some purposes, for example, when thepharmaceutical composition is meant for infusion or injection, it isoften desirable that the composition comprises a buffer, which iscapable of buffering a solution to a pH in the range of 4 to 10, such as5 to 9, for example 6 to 8.

The buffer may for example be selected from the group consisting ofTris, acetate, glutamate, lactate, maleate, tartrate, phosphate,citrate, carbonate, glycinate, L-histidine, glycine, succinate andtriethanolamine buffer.

The buffer may furthermore for example be selected from USP compatiblebuffers for parenteral use, in particular, when the pharmaceuticalformulation is for parenteral use. For example the buffer may beselected from the group consisting of monobasic acids such as acetic,benzoic, gluconic, glyceric and lactic; dibasic acids such as aconitic,adipic, ascorbic, carbonic, glutamic, malic, succinic and tartaric,polybasic acids such as citric and phosphoric; and bases such asammonia, diethanolamine, glycine, triethanolamine, and Tris.

Parenteral vehicles (for subcutaneous, intravenous, intraarterial, orintramuscular injection) include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's and fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Examples are sterile liquids such as water and oils, with orwithout the addition of a surfactant and other pharmaceuticallyacceptable adjuvants. In general, water, saline, aqueous dextrose andrelated sugar solutions, glycols such as propylene glycols orpolyethylene glycol, Polysorbate 80 (PS-80), Polysorbate 20 (PS-20), andPoloxamer 188 (P188) are preferred liquid carriers, particularly forinjectable solutions. Examples of oils are those of animal, vegetable,or synthetic origin, for example, peanut oil, soybean oil, olive oil,sunflower oil, fish-liver oil, another marine oil, or a lipid from milkor eggs.

The formulations may also contain a surfactant. Preferred surfactantsinclude, but are not limited to: the polyoxyethylene sorbitan esterssurfactants (commonly referred to as the Tweens), especially PS-20 andPS-80; copolymers of ethylene oxide (EO), propylene oxide (PO), and/orbutylene oxide (BO), sold under the DOWFAX™ tradename, such as linearEO/PO block copolymers; octoxynols, which can vary in the number ofrepeating ethoxy (oxy-1,2-ethanediyl) groups, with octoxynol-9 (TritonX-100, or t-octylphenoxypolyethoxyethanol) being of particular interest;(octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipidssuch as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such asthe Tergitol™ NP series; polyoxyethylene fatty ethers derived fromlauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants),such as triethyleneglycol monolauryl ether (Brij 30); and sorbitanesters (commonly known as the SPANs), such as sorbitan trioleate (Span85) and sorbitan monolaurate. A preferred surfactant for including inthe emulsion is PS-20 or PS-80.

Mixtures of surfactants can be used, e.g. PS-80/Span 85 mixtures. Acombination of a polyoxyethylene sorbitan ester such as polyoxyethylenesorbitan monooleate (PS-80) and an octoxynol such ast-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable. Anotheruseful combination comprises laureth 9 plus a polyoxyethylene sorbitanester and/or an octoxynol.

Preferred amounts of surfactants are: polyoxyethylene sorbitan esters(such as PS-80) 0.01 to 1% w/v, in particular about 0.1% w/v; octyl- ornonylphenoxy polyoxyethanols (such as Triton X-100, or other detergentsin the Triton series) 0.001 to 0.1% w/v, in particular 0.005 to 0.02%w/v; polyoxyethylene ethers (such as laureth 9) 0.1 to 20% w/v,preferably 0.1 to 10% w/v and in particular 0.1 to 1% w/v or about 0.5%w/v.

In certain embodiments, the composition consists essentially ofL-histidine (20 mM), saline (150 mM) and 0.2% w/v PS-20 at a pH of 5.8with 250 □g/mL of APA (Aluminum Phosphate Adjuvant). PS-20 can rangefrom 0.005 to 0.1% w/v with the presence of PS-20 or PS-80 informulation controlling aggregation during simulated manufacture and inshipping using primary packaging. Process consists of combining blend ofup to 44 S. pneumoniae polysaccharide serotypes in L-histidine, sodiumchloride, and PS-20 then combining this blended material with APA andsodium chloride with or without antimicrobial preservatives.

The choice of surfactant may need to be optimized for different drugproducts and drug substances. For multivalent vaccines containing 15 ormore S. pneumoniae polysaccharide serotypes, PS-20 and P188 arepreferred. The choice of chemistry used to prepare the conjugate canalso influence the stabilization of the formulation. In particular, asexemplified below, pneumococcal polysaccharide-protein conjugatesprepared in aqueous or DMSO solvent and combined in a multivalentcomposition show significant differences in stability depending on theparticular surfactant systems used for formulation.

For the formulations described herein, a poloxamer generally has amolecular weight in the range from 1,100 Da to 17,400 Da, from 7,500 Dato 15,000 Da, or from 7,500 Da to 10,000 Da. The poloxamer can beselected from poloxamer 188 or poloxamer 407. The final concentration ofthe poloxamer in the formulations of the invention is from 0.001 to 5%w/v, or 0.025 to 1% w/v. A surfactant system comprising a poloxamer mustfurther comprise a polyol. In certain aspects, the polyol is propyleneglycol and is at final concentration from 1 to 20% w/v. In certainaspects, the polyol is polyethylene glycol 400 and is at finalconcentration from 1 to 20% w/v.

Suitable polyols for the formulations are polymeric polyols,particularly polyether diols including, but are not limited to,propylene glycol and polyethylene glycol, Polyethylene glycol monomethylethers. Propylene glycol is available in a range of molecular weights ofthe monomer from ˜425 Da to ˜2,700 Da. Polyethylene glycol andPolyethylene glycol monomethyl ether is also available in a range ofmolecular weights ranging from ˜200 Da to ˜35,000 Da including but notlimited to PEG200, PEG300, PEG400, PEG1000, PEG MME 550, PEG MME 600,PEG MME 2000, PEG MME 3350 and PEG MME 4000. A preferred polyethyleneglycol is polyethylene glycol 400. The final concentration of the polyolin the formulations may be 1 to 20% w/v or 6 to 20% w/v.

The formulation also contains a pH-buffered saline solution. The buffermay, for example, be selected from the group consisting of Tris,acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate,carbonate, glycinate, L-histidine, glycine, succinate, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS(3-(N-morpholino)propanesulfonic acid), MES(2-(N-morpholino)ethanesulfonic acid) and triethanolamine buffer. Thebuffer is capable of buffering a solution to a pH in the range of 4 to10, 5.2 to 7.5, or 5.8 to 7.0. In certain aspects, the buffer selectedfrom the group consisting of phosphate, succinate, L-histidine, MES,MOPS, HEPES, acetate or citrate. The buffer may furthermore, forexample, be selected from USP compatible buffers for parenteral use, inparticular, when the pharmaceutical formulation is for parenteral use.The concentrations of buffer will range from 1 mM to 50 mM or 5 mM to 50mM. In certain aspects, the buffer is L-histidine at a finalconcentration of 5 mM to 50 mM, or succinate at a final concentration of1 mM to 10 mM. In certain aspects, the L-histidine is at a finalconcentration of 20 mM±2 mM.

While the saline solution (i.e., a solution containing NaCl) ispreferred, other salts suitable for formulation include but are notlimited to, CaCl₂, KCl and MgCl₂ and combinations thereof. Non-ionicisotonic agents including but not limited to sucrose, trehalose,mannitol, sorbitol and glycerol may be used in lieu of a salt. Suitablesalt ranges include, but not are limited to 25 mM to 500 mM or 40 mM to170 mM. In one aspect, the saline is NaCl, optionally present at aconcentration from 20 mM to 170 mM.

In a preferred embodiment, the formulations comprise a L-histidinebuffer with sodium chloride.

In another embodiment, the pharmaceutical composition is delivered in acontrolled release system. For example, the agent can be administeredusing intravenous infusion, a transdermal patch, liposomes, or othermodes of administration. In another embodiment, polymeric materials areused; e.g. in microspheres in or an implant.

The compositions described herein may also include one or more proteinsfrom S. pneumoniae. Examples of S. pneumoniae proteins suitable forinclusion include those identified in International Patent ApplicationPublication Nos. WO 02/083855 and WO 02/053761.

Analytical Methods Molecular Weight and Concentration Analysis ofConjugates Using HPSEC/UV/MALS/RI Assay

Conjugate samples are injected and separated by high performancesize-exclusion chromatography (HPSEC). Detection is accomplished withultraviolet (UV), multi-angle light scattering (MALS) and refractiveindex (RI) detectors in series. Protein concentration is calculated fromUV280 using an extinction coefficient. Polysaccharide concentration isdeconvoluted from the RI signal (contributed by both protein andpolysaccharide) using the do/dc factors which are the change in asolution's refractive index with a change in the solute concentrationreported in mL/g. Average molecular weight of the samples are calculatedby Astra software (Wyatt Technology Corporation, Santa Barbara, Calif.)using the measured concentration and light scattering information acrossthe entire sample peak. There are multiple forms of average values ofmolecular weight for polydispersed molecules. For example,number-average molecular weight Mn, weight-average molecular weight Mw,and z-average molecular weight Mz (Molecules, 2015, 20:10313-10341).Unless specified, the term “molecular weight”, as used throughout thespecification, is the weight-average molecular weight.

Determination of Lysine Consumption in Conjugated Protein as a Measureof the Number of Covalent Attachments Between Polysaccharide and CarrierProtein

The Waters AccQ-Tag amino acid analysis (AAA) is used to measure theextent of conjugation in conjugate samples. Samples are hydrolyzed usingvapor phase acid hydrolysis in the Eldex workstation, to break thecarrier proteins down into their component amino acids. The free aminoacids are derivatized using 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC). The derivatized samples are then analyzed using UPLCwith UV detection on a C18 column. The average protein concentration isobtained using representative amino acids other than lysine. Lysineconsumption during conjugation (i.e., lysine loss) is determined by thedifference between the average measured amount of lysine in theconjugate and the expected amount of lysine in the starting protein.

Free Polysaccharide Testing

Free polysaccharide (i.e., polysaccharide that is not conjugated withCRM197) in the conjugate sample is measured by first precipitating freeprotein and conjugates with deoxycholate (DOC) and hydrochloric acid.Precipitates are then filtered out and the filtrates are analyzed forfree polysaccharide concentration by HPSEC/UV/MALS/RI. Freepolysaccharide is calculated as a percentage of total polysaccharidemeasured by HPSEC/UV/MALS/RI.

Free Protein Testing

Free polysaccharide, polysaccharide-CRM197 conjugate, and free CRM197 inthe conjugate samples are separated by capillary electrophoresis inmicellar electrokinetic chromatography (MEKC) mode. Briefly, samples aremixed with MEKC running buffer containing 25 mM borate, 100 mM SDS, pH9.3, and are separated in a preconditioned bare-fused silica capillary.Separation is monitored at 200 nm and free CRM197 is quantified with aCRM197 standard curve. Free protein results are reported as a percentageof total protein content determined by the HPSEC/UV/MALS/RI procedure.

Having described various embodiments of the invention with reference tothe accompanying description and drawings, it is to be understood thatthe invention is not limited to those precise embodiments, and thatvarious changes and modifications may be effected therein by one skilledin the art without departing from the scope or spirit of the inventionas defined in the appended claims.

The following examples illustrate, but do not limit the invention.

EXAMPLES Example 1: Preparation of S. pneumoniae CapsularPolysaccharides

Methods of culturing pneumococci are well known in the art. See, e.g.,Chase, 1967, Methods of Immunology and Immunochemistry 1:52. Methods ofpreparing pneumococcal capsular polysaccharides are also well known inthe art. See, e.g., European Patent No. EP 0 497 524 B 1. The processdescribed below generally follows the method described in EuropeanPatent No. EP 0 497 524 B1 and is generally applicable to allpneumococcal serotypes except where specifically modified.

Isolates of pneumococcal serotypes 3, 8, 12F were obtained fromUniversity of Pennsylvania (Dr. Robert Austrian). Isolates ofpneumococcal serotypes 15A, 16F, 23A, 24F, 35B were obtained from theMerck Culture Collection. Isolates of pneumococcal serotype 23B and 31were obtained from Centers for Disease Control and Prevention (Atlanta,Ga.). Isolate of pneumococcal serotype 17F was obtained from the FDAOffice of Biologics (Dr. John Robbins). Isolate of pneumococcal serotype20 was obtained from ATCC. Where needed, subtypes can be differentiatedon the basis of Quelling reaction using specific antisera. See, e.g.,U.S. Pat. No. 5,847,112. The obtained isolates were further clonallyisolated by plating serially in two stages on agar plates consisting ofan animal-component free medium containing soy peptone, yeast extract,and glucose without hemin. Clonal isolates for each serotype werefurther expanded in liquid culture using animal-component free mediacontaining soy peptone, yeast extract, HEPES, sodium chloride, sodiumbicarbonate, potassium phosphate, glucose, and glycerol to prepare thepre-master cell banks.

The production of each serotype of pneumococcal polysaccharide consistedof a cell expansion and batch production fermentation followed bychemical inactivation prior to downstream purification. A thawed cellbank vial from each serotype was expanded using a shake flask or culturebottle containing a pre-sterilized animal-component free growth mediacontaining soy peptone or soy peptone ultrafiltrate, yeast extract oryeast extract ultrafiltrate, HEPES, sodium chloride, sodium bicarbonate,potassium phosphate, and glucose. The cell expansion culture was grownin a sealed shake flask or bottle to minimize gas exchange withtemperature and agitation control. After achieving a specified culturedensity, as measured by optical density at 600 nm, a portion of the cellexpansion culture was transferred to a production fermentor containingpre-sterilized animal-component free growth media containing soy peptoneor soy peptone ultrafiltrate, yeast extract or yeast extractultrafiltrate, sodium chloride, potassium phosphate, and glucose.Temperature, pH, pressure, and agitation were controlled. Airflowoverlay was also controlled as sparging was not used.

The batch fermentation was terminated via the addition of a chemicalinactivating agent, phenol, when glucose was nearly exhausted. Purephenol was added to a final concentration of 0.8-1.2% to inactivate thecells and liberate the capsular polysaccharide from the cell wall.Primary inactivation occurs for a specified time within the fermentorwhere temperature and agitation continue are to be controlled. Afterprimary inactivation, the batch was transferred to another vessel whereit was held for an additional specified time at controlled temperatureand agitation for complete inactivation. This was confirmed by eithermicrobial plating techniques or by verification of the phenolconcentration and specified time. The inactivated broth was thenpurified.

Purification of Ps

The purification of the pneumococcal polysaccharide consisted of severalcentrifugation, depth filtration, concentration/diafiltrationoperations, and precipitation steps. All procedures were performed atroom temperature unless otherwise specified.

Inactivated broth from the fermentor cultures of S. pneumoniae wereflocculated with a cationic polymer (such as BPA-1000, Petrolite“Tretolite” and “Spectrum 8160” and poly(ethyleneimine), “MilliporepDADMAC”). The cationic polymers bound to the impurity protein, nucleicacids and cell debris. Following the flocculation step and an agingperiod, flocculated solids were removed via centrifugation and multipledepth filtration steps. Clarified broth was concentrated and diafilteredusing a 100 kDa to 500 kDa MWCO (molecular weight cutoff) filter.Diafiltration was accomplished using Tris, MgCl₂ buffer and sodiumphosphate buffer. Diafiltration removed residual nucleic acid andprotein.

Further impurities removal was accomplished by reprecipitation of thepolysaccharide in sodium acetate and phenol with denatured alcoholand/or isopropanol. During the phenol precipitation step, sodium acetatein sodium phosphate saline buffer and phenol (liquefied phenols or solidphenols) was charged to the diafiltered retentate. Alcohol fractionationof the polysaccharide was then conducted in two stages. In the firststage a low percent alcohol was added to the preparation to precipitatecellular debris and other unwanted impurities, while the crudepolysaccharide remained in solution. The impurities were removed via adepth filtration step. The polysaccharide was then recovered from thesolution by adding additional isopropanol or denatured alcohol to thebatch. The precipitated polysaccharide pellet was recovered bycentrifugation, triturated and dried as a powder and stored frozen at−70° C.

Example 2: General Conjugation Methods Polysaccharide Size Reduction andOxidation

Purified pneumococcal capsular polysaccharide powder was dissolved inwater and 0.45-micron filtered. Unless otherwise specified,polysaccharides were homogenized to reduce the polysaccharide molecularmass. Homogenization pressure and number of passes through thehomogenizer were controlled to serotype-specific targets (150-1000 bar;4-7 passes).

Size-reduced polysaccharide was 0.2 micron filtered and thenconcentrated and diafiltered against distilled water using a 5 kDa or 10kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation.

The purified polysaccharides were prepared for conjugation, i.e.,activated, using sodium metaperiodate oxidation (See Anderson et al.,1986, J. Immunol. 137:1181-1186; and U.S. Patent Application PublicationNo. US20110195086). A 100 mM sodium metaperiodate solution was added tothe polysaccharide solution in 50 mM sodium acetate. The amount ofsodium metaperiodate added was serotype-specific, ranging fromapproximately 0.1 to 0.5 moles of sodium metaperiodate per mole ofpolysaccharide repeating unit, to achieve a target level ofpolysaccharide activation (moles aldehyde per mole of polysacchariderepeating unit). The sample was mixed for a target incubation timeprotected from light.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by distilled water using a 5 kDa or 10 kDa NMWCOtangential flow ultrafiltration membrane, followed by additionaldiafiltration against water. Ultrafiltration for all serotypes wasconducted at 2-8° C.

Conjugation

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 1-6 mgPs/mL with sucrose concentration of 0.5-30% w/v. CRM197 was formulatedfor lyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide and CRM197 solutions wereblended to achieve a target polysaccharide concentration and apolysaccharide to CRM197 mass ratio. The mass ratio was selected tocontrol the polysaccharide to CRM197 ratio in the resulting conjugate.Sodium cyanoborohydride (1 mole per mole of polysaccharide repeatingunit) was added, and conjugation proceeded for a target incubation timeat 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 mole per mole of polysaccharide repeating unit)was added following the conjugation reaction. The batch was diluted into150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20,at approximately 4° C. Potassium phosphate buffer was then added toneutralize the pH.

Final Filtration and Product Storage

Conjugates were then dialyzed against 150 mM sodium chloride with 0.05%(w/v) polysorbate 20 at approximately 4° C. using a 300 kDa NMWCmembrane, or diafiltered against 150 mM sodium chloride, with or without25 mM potassium phosphate pH 7 using a 30 kDa NMWC tangential flowultrafiltration membrane, followed by concentration and diafiltrationagainst 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015%(w/v) polysorbate 20, at 4° C. using a 300 kDa NMWCO tangential flowultrafiltration membrane. The retentate batch was diluted withadditional 10 mM histidine in 150 mM sodium chloride, pH 7.0 and 0.2micron filtered. The final conjugate solution was dispensed intoaliquots and frozen at ≤−60° C.

Example 3: Acid Hydrolysis of Polysaccharides from Serotypes 12F, 23A,24F, and 31

Conjugation of pneumococcal polysaccharides to proteins by reductiveamination in an aprotic solvent such as DMSO has been previouslydescribed. Activated polysaccharides (Ps) and proteins (Pr) aretypically lyophilized, resuspended in DMSO, then blended and incubatedwith sodium cyanoborohydride and sodium borohydride to achieveconjugation. Polysaccharides may be mechanically size-reduced (e.g. byhomogenization) prior to oxidation to reduce the Ps molecular mass andprovide a consistent Ps size for conjugation. For many pneumococcalserotypes, conjugation of mechanically size-reduced and oxidized Psyields conjugates that meet target attributes for size, lysineconsumption, free polysaccharide, and free protein. However for someserotypes it was found that target conjugate attributes were difficultto achieve with this process, even after optimizing process parameters.

Ps Size Reduction

Purified pneumococcal capsular polysaccharide powder from serotypes 23A,24F, and 31 were dissolved in water. Different experimental arms wereprocessed either mechanically by homogenization or chemically by acidhydrolysis to reduce the molecular mass of the Ps. Homogenizationpressure and number of passes through the homogenizer were controlled toserotype-specific targets (150-1000 bar; 4-7 passes). Acid hydrolysiswas performed by heating the batch to 90-92° C., adding concentratedacetic acid to a final concentration of 200 mM, then incubating for upto 90 minutes. At the end of the incubation period, the batch wasneutralized by adding concentrated potassium phosphate pH 7 buffer to afinal concentration of 400 mM and cooling to ≤22° C. Size-reducedpolysaccharide was 0.2-micron filtered and then concentrated anddiafiltered against water using a 5 kDa or 10 kDa NMWC tangential flowultrafiltration membrane.

The polysaccharides were conjugated as described in Example 2.

Experimental conditions and results are summarized in Table 1.

TABLE 1 Summary of experimental arms, Ps size-reduction byhomogenization vs acid hydrolysis, for S. pneumoniae serotypes 23A, 24F,and 31 Conjugation Conditions, [Ps] Conjugate Size Size NaIO₄ Oxidized(mg/mL)/Ps:Pr/ Conjugate Free Ps/ Reduction Reduction Charge Ps Mw Time(hrs) / Conjugate Lys Loss Free Pr Serotype Method Conditions (MEq) (kD)[NaCl] (mM) Mw (kD) (mol/mol) Fraction 23A Homogenized 600 bar/ 0.20 3193/1.5/3/25 6774 8.4 7%/ 3 passes 18% Acid 90° C./ 0.20 97 3/1.5/2/252996 12.6 11%/ Hydrolysis 90 min <3% 24F Homogenized 600 bar/ 0.18 2272/1.5/17/ 8727 10.6 51%/ 5 passes 25 15% Acid 92° C./ 0.18 1002/1.5/15/25 5816 9.0 22%/ Hydrolysis 90 min 2% 31 Homogenized 400 bar/0.12 186 4/1.5/4/25 3323 11.5 18%/ 5 passes 3% Acid 90° C./ 0.16 1194/.2/4/25 3201 13.3 2%/ hydrolysis 30 min <2%

As seen in Table 1, size reduction by acid hydrolysis provided a meansto achieve higher lysine loss (serotype 23A), lower free Ps (serotype24F and 31), or lower free Pr (serotypes 23A, 24F and 31) compared tohomogenization. For serotypes 23A and 24F, the higher free proteinlevels for homogenization may have been associated with aggregated formswhich may in turn have contributed to the higher measured conjugate Mwlevels.

Without being bound by any particular theory, the data suggest that thelower molecular weight of oxidized polysaccharide (preferentially lessthan 150 KDa) achieved through acid hydrolysis helped to improve theconjugation (less free Ps or free Pr). It is believed that alternativesize reduction processes (e.g., through acid hydrolysis) may be used toachieve polysaccharide at this preferred size range to achieve similarconjugation benefits.

Impact of Acid Type while Maintaining Constant pH on Acid Hydrolysis ofSerotype 12F

To determine whether the acid type had an impact on polysaccharide size,acid hydrolysis using hydrochloric acid was compared to acetic acid.Purified pneumococcal capsular polysaccharide powder from S. pneumoniaeserotype 12F was dissolved in water and 0.45 μm filtered. The batch wasdiluted to 2.5 g Ps/L and split into two arms. Acid hydrolysis wasperformed on both arms by first heating them to 80° C. Acid was added tomaintain similar pH across both arms. To one arm, glacial acetic acidwas added to a final concentration of 200 mM, pH 2.6. To the other arm,IN hydrochloric acid was added to a final concentration of 2.5 mM, pH2.7. Both arms were then incubated for 155 minutes. At the end of theincubation period, the arms were neutralized by adding concentratedpotassium phosphate pH 7 buffer to a final concentration ofapproximately 400 mM and cooling to 4° C. The results are shown in Table2.

TABLE 2 Acid hydrolysis of S. pneumoniae 12F polysaccharide usinghydrochloric acid Acid Acid hydrolysis hydrolysis Acid temp timehydrolysis Mn Mw Ps Conc Description (° C.) (min) pH (kD) (kD)Polydispersity (mg/mL) Acid N/A N/A N/A 349.7 448.6 1.283 2.496hydrolysis feed Acid 80.0 +/− 155 2.64 75.2 104.4 1.388 1.866 hydrolyzed0.5 with 200 mM acetic acid Acid 80.0 +/− 155 2.70 76.5 106.6 1.3931.856 hydrolyzed 0.5 with 2.5 mM hydrochloric acid

Both acid hydrolysis conditions produced similar sized polysaccharides.

Example 4: Impact of Sucrose to Polysaccharide Mass Ratio on Dissolutionof Polysaccharides from S. pneumoniae Serotypes 3, 8 and 24F in DMSO

In preparation for conjugation of polysaccharides to proteins in anaprotic solvent, polysaccharide and protein solutions are typicallylyophilized. For most pneumococcal serotypes, formulating activatedpolysaccharides (Ps) for lyophilization in aqueous solution at 6 mgPs/mL with sucrose concentration of 5% w/v (50 mg sucrose/mL) resultedin lyophilized material suitable for redissolution in DMSO andconjugation. For serotypes 3, 8, and 24F, it was discovered that thisformulation (6 mg Ps/mL, 50 mg sucrose/mL, sucrose:Ps mass ratio=8.3)yielded lyophilized material that did not dissolve in DMSO.

Experiments were performed to optimize the activated polysaccharidelyophilization formulation for dissolution in DMSO. Activatedpolysaccharides were formulated across a range of polysaccharideconcentrations (1-6 mg Ps/mL) and sucrose concentrations (50-300 mgsucrose/mL) in polypropylene containers. Solutions were lyophilized toremove water, then DMSO was added at ambient temperature with mixing toredissolve polysaccharides. Successful dissolution was determined byvisual observation.

Results for serotype 3, 24F, and 8 are shown in Tables 3, 4, and 5respectively.

TABLE 3 Serotype 3 Lyophilization Formulation and DissolutionExperiments Post-lyo addition of Pre-lyo formulation conditions DMSOExperimental Polysaccharide [Ps], [sucrose], Sucrose:Ps [Ps], ConditionMw (kD) (mg/mL) (mg/mL) mass ratio mg/mL Dissolution 1 211 6.0 30 5.02.0 No 2 211 6.0 40 6.7 4.0 No 3 211 6.0 50 8.3 6.0 No 4 212 1.0 5.0 5.02.0 No 5 212 1.0 10 10 2.0 No 6 212 1.0 20 20 2.0 No 7 212 1.0 50 50 2.0Yes 8 212 1.0 100 100 2.0 Yes 9 212 6.0 50 8.3 6.0 No 10 212 4.0 200 506.0 Yes 11 212 3.0 150 50 6.0 Yes 12 212 2.0 100 50 6.0 Yes 13 212 4.0200 50 3.0 Yes 14 257 2.0 100 50 2.0-5.0 Yes (all (multiple arms) arms)15 253 2.0 40 20 3.0 No 16 253 2.0 60 30 3.0 Yes 17 253 2.0 80 40 3.0Yes 18 253 2.0 100 50 3.0 Yes 19 253 4.0 80 20 3.0 No 20 253 4.0 120 303.0 Not fully dissolved 21 253 4.0 160 40 3.0 Yes 22 253 4.0 200 50 3.0Yes

TABLE 4 Serotype 24F Lyophilization Formulation and DissolutionExperiments Post-lyo addition of Pre-lyo formulation conditions DMSOExperimental Polysaccharide [Ps], [sucrose], Sucrose:Ps [Ps], ConditionMw (kD) (mg/mL) (mg/mL) mass ratio mg/mL Dissolution 1 142 6 50 8.32.0-8.0 No (multiple arms) 2 142 6 50 8.3 4.0 No 3 142 6 300 50 6.0 Yes4 142 4 200 50 4.0 Yes* 5 142 3 150 50 4.0 Yes 6 142 2 100 50 4.0 Yes 7142 2 100 50 4.0-8.0 Yes (multiple arms) 8 132 6 50 8.3 4.0-8.0 No(multiple arms) 9 132 2 100 50 4.0-8.0 Yes (multiple arms) 10 142 2 10050 3.0-4.0 Yes (multiple arms) 11 227 2 100 50 2.0-6.0 Yes (multiplearms) 12 63 6 50 8.3 3.0-4.0 No (multiple arms) 13 63 2 100 50 3.0-5.0Yes (multiple arms) *In Experimental condition 4, a single small visibleparticle was observed that it is not believed to be due to incompletedissolution

TABLE 5 Serotype 8 Lyophilization Formulation and DissolutionExperiments Pre-lyo formulation conditions Post-lyo additionExperimental Polysaccharide [Ps], [sucrose] Sucrose:Ps of DMSO ConditionMw (kD) (mg/mL) (mg/mL) mass ratio [Ps], mg/mL Dissolution 1 233 6.0 508.3 4.0-8.0 No (multiple arms) 2 233 2.0 100 50 4.0-8.0 Yes (multiplearms) 3 252 2.0 100 50 4.0-8.0 Yes (multiple arms)

For these serotypes, full dissolution was observed across the range ofpolysaccharide concentrations studied (for both lyophilization anddissolution), however dissolution was dependent on both thepolysaccharide and sucrose concentrations. Specifically, when the massratio of sucrose was less than 30× that of the polysaccharide,dissolution in DMSO following lyophilization was not achieved. The mostconsistent dissolution results were found when the sucrose mass ratiowas at least 40× that of the polysaccharide.

Several arms from the positive dissolution conditions were successfullyconjugated to CRM197 as described in Example 2.

Impact of Sugar Type and Sugar to Polysaccharide Mass Ratio onDissolution of S. pneumoniae Polysaccharide from Serotype 3 in DMSO

Experiments were performed to assess the impact of sugar type andsugar:polysaccharide mass ratio on lyophilized activated polysaccharidedissolution in DMSO. Activated serotype 3 polysaccharide was formulatedat either 2.5 or 6 mg Ps/mL and at a range of sugar concentrations(50-150 mg sugar/mL) in polypropylene containers. Sugars tested weresucrose, trehalose and mannitol. Solutions were lyophilized to removewater then DMSO was added at ambient temperature with mixing toredissolve polysaccharides. Successful dissolution was determined byvisual observation. The results are shown in Table 6.

TABLE 6 Impact of Sugar Type on Serotype 3 Polysaccharide Pre-lyoformulation conditions Post-lyo addition Sugar:Ps of DMSO ExperimentalPolysaccharide [Ps], Sugar [Sugar], mass [Ps], Condition Mw (kD) (mg/mL)Type (mg/mL) ratio mg/mL Dissolution A1 171 6.0 Sucrose 50 8.3 4.4 No A2171 2.5 Sucrose 50 20 4.4 No A3 171 2.5 Sucrose 75 30 4.4 Yes A4 171 2.5Sucrose 100 40 4.4 Yes A5 171 2.5 Sucrose 125 50 4.4 Yes A6 171 2.5Sucrose 150 60 4.4 Yes B1 171 6.0 Trehalose 50 8.3 4.4 No B2 171 2.5Trehalose 50 20 4.4 No B3 171 2.5 Trehalose 75 30 4.4 Yes B4 171 2.5Trehalose 100 40 4.4 Yes B5 171 2.5 Trehalose 125 50 4.4 Yes B6 171 2.5Trehalose 150 60 4.4 Yes C1 171 6.0 Mannitol 50 8.3 4.4 No C2 171 2.5Mannitol 50 20 4.4 No C3 171 2.5 Mannitol 75 30 4.4 Yes* C4 171 2.5Mannitol 100 40 4.4 Yes C5 171 2.5 Mannitol 125 50 4.4 Yes C6 171 2.5Mannitol 150 60 4.4 No *viscous

For all sugars used, dissolution in DMSO was achieved for sugar massratios of 30× and higher with the exception of mannitol, which appearedto reach the solubility limit in DMSO at 60×. The most consistentdissolution results were found when the sugar mass ratio was at least40× that of the polysaccharide.

Another experiment was performed looking at using combinations of sugarsin activated serotype 3 polysaccharide lyophilization formulations. Allarms were formulated to a total sugar mass ratio at 40× since this ratioyielded consistent dissolution in DMSO. The molecular weight of thepolysaccaride was 171 kD. All arms used sucrose, either alone, withtrehalose or with mannitol. Solutions were lyophilized to remove waterthen DMSO was added at ambient temperature with mixing to redissolvepolysaccharides. Successful dissolution was determined by visualobservation. The results are shown in Table 7.

TABLE 7 Sugar combinations for dissolation of polysaccharides in DMSOPre-lyo formulation conditions Total Post-lyo addition Sucrose:Ps Sugar2:Ps Sugar:Ps of DMSO [Ps], [Sucrose] mass Sugar 2 [Sugar 2] mass mass[Ps], (mg/mL) [(mg/mL) ratio Type (mg/mL) ratio ratio mg/mL Dissolution2.5 100 40 N/A 0 0 40 4.4 Yes 2.5 75 30 Trehalose 25 10 40 4.4 Yes 2.550 20 Trehalose 50 20 40 4.4 Yes 2.5 75 30 Mannitol 25 10 40 4.4 Yes

Dissolution in DMSO was achieved for all arms tested.

Example 5: Effect of Sodium Chloride on Conjugation of S. pneumoniaeSerotypes 15A, 16F, 17F, 20, 24F, and 35B Using Reductive Amination inDMSO

Activated polysaccharides were formulated for lyophilization at 2-6 mgPs/mL with sucrose concentrations of 5-10% w/v in polypropylenecontainers. CRM197 was formulated for lyophilization at 6 mg Pr/mL withsucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved in DMSO. For somearms, a 5 M stock solution of sodium chloride was used to spike theCRM197 solution prior to lyophilization or the redissolved Ps solutionto achieve final concentrations during conjugation of 10-100 mM sodiumchloride. Other process parameters were maintained constant in theseexperiments.

Redissolved Ps and CRM197 solutions were blended and mixed to targetserotype-specific polysaccharide and protein concentrations. Sodiumcyanoborohydride (1 moles per mole of polysaccharide repeating unit) wasadded, and conjugation proceeded for a serotype-specific duration.Reduction with sodium borohydride and final filtration were performed asdescribed in Example 2.

Results

Experimental results for polysaccharide from S. pneumoniae serotype 20showing the impact of sodium chloride during conjugation on conjugateattributes are shown in FIGS. 1, 2, and 3. Experimental results for S.pneumoniae serotypes 16F and 24F are shown in Table 8.

As shown in FIG. 1 for serotype 20, increasing sodium chlorideconcentration during conjugation (up to ca. 50 mM) increased conjugatesize. As shown in FIG. 2, increasing sodium chloride concentration (upto ca. 25 mM) increased lysine consumption, and as shown in FIG. 3,increasing sodium chloride concentration (up to ca. 50 mM) decreasedfree Ps and free Pr.

TABLE 8 Impact of sodium chloride on conjugate attributes for S.pneumoniae serotypes 16F and 24F NaCl Concentration During ConjugateLysine Conjugation Mn/Mw Consumption Free Ps Free Pr Serotype (mM) (kD)(mol/mol) Fraction Fraction 16F 0  438/1040 9.3 23%  7% 25 1161/394411.0  7%  4% 24F 0 2534/6478 1.8 85% 24% 25 2059/3402 4.5 59% 10% 15A 0 954/1343 7.8 51% 23% 25 1748/3371 8.9 23%  7% 35B 0 N/R >32%   25 32% 5%

As shown in Table 8 for serotype 16F, inclusion of 25 mM sodium chlorideduring conjugation increased conjugate size and lysine consumption,while decreasing free Ps and free Pr. As shown in Table 8 for serotype24F, inclusion of 25 mM sodium chloride during conjugation increasedlysine consumption, while decreasing free Ps and free Pr.

Similar results were found for serotypes 15A and 35B. For serotype 15A,inclusion of 25 mM sodium chloride during conjugation increasedconjugate size and lysine consumption, while decreasing freepolysaccharide and free protein compared to no salt during conjugation.For serotype 35B, inclusion of 25 mM sodium chloride during conjugationincreased conjugate size and lysine consumption (data not shown), whiledecreasing free protein compared to no salt during conjugation.

Effect of Salt Type and Concentration on Conjugation of S. pneumoniaeSerotype 17F Using Reductive Amination in DMSO

Activated serotype 17F polysaccharide was formulated for lyophilizationat 6 mg Ps/mL with sucrose concentrations of 5% w/v in polypropylenecontainers. CRM197 was formulated for lyophilization at 6 mg Pr/mL withsucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved in DMSO. For somearms, concentrated salt solutions were spiked into the redissolved Pssolution or the Ps-CRM blend to achieve final concentrations duringconjugation of 1-100 mM salt. Stock solutions used included 1M or 5Msodium chloride, 1M or 3M potassium chloride and 1M magnesium chloride.Other process parameters were maintained constant in these experiments.

Redissolved Ps and CRM197 solutions were blended and mixed to 2.7 gPs/mL and 1.8 mg CRM197/mL. Sodium cyanoborohydride (1 moles per mole ofpolysaccharide repeating unit) was added, and conjugation proceeded for1 hour. Sodium borohydride (2 mole per mole of polysaccharide repeatingunit) was added following the conjugation reaction. The batch wasdiluted into 150 mM sodium chloride with approximately 0.025% (w/v)polysorbate 20, at approximately 4° C. Potassium phosphate buffer wasthen added to neutralize the pH.

Conjugates were then dialyzed against 150 mM sodium chloride with 0.05%(w/v) polysorbate 20 at approximately 4° C. using a 300 kDa NMWCmembrane. The final conjugate solution was dispensed into aliquots andmaintained at 4° C.

Results

The results are shown in Table 9. 2% is the limit of detection. LysineSalt Conc. Conjugate Conjugate % Consumption % (mM) Salt Type Mn (kD) Mw(Kd) Free Ps (mol/mol) Free Protein 0 N/A 966 1419 8% 6.5 7% 1 NaCl 10411671 10%  8.1 6% 2 NaCl 1318 2113 10%  7.8 5% 5 NaCl 1964 2933 5% 8.8 3%10 NaCl 2407 3640 8% 9.4 2% 12.5 NaCl 2529 3749 7% 9.6 <2%  25 NaCl 26573772 4% 8.8 4% 50 NaCl 3036 4198 3% 9.5 <2%  100 NaCl 3031 4249 3% 9.4<2%  0 N/A 1012 1339 9% 6.8 7% 1 KCl 1189 1653 9% 7.0 6% 2 KCl 1533 20185% 7.5 4% 5 KCl 1862 2474 4% 8.7 3% 10 KCl 2416 3278 6% 9.1 3% 12.5 KCl2654 3532 4% 9.2 2% 25 KCl 2641 3446 3% 9.6 3% 0 N/A 900 1335 9% 6.3 7%25 MgCl2 2878 4361 9% 8.2 16%  50 MgCl2 1374 1994 12%  6.6 18%  100MgCl2 798 1175 29%  4.0 34% 

For sodium chloride and potassium chloride conditions, increasing saltconcentration during conjugation increased conjugate size and lysineconsumption and decreased free Ps and free Pr. These effects plateauedat approximately 12.5 mM for both salt types. 25 mM and 50 mM magnesiumchloride showed an increase in conjugate size and lysine consumptioncompared to the no salt condition. However, there seems to be increasedfree polysaccharide and free protein levels, and decreased extent ofconjugation (measured by lysine loss and conjugate size) with increasingconcentration of magnesium chloride from 25 mM to 100 mM. Therefore, itmay be preferred to keep magnesium chloride in the concentration rangeof 0-50 mM.

Example 6: Formulation of Monovalent Conjugates

Pneumococcal polysaccharide-CRM197 conjugates were prepared as describedin Examples 2-5. The required volume of bulk conjugates needed to obtainthe target concentration of individual serotypes were calculated basedon batch volume and concentration of individual bulk polysaccharideconcentrations. Individual serotypes (12F, 15A, 16F, 17F, 23A, 23B, 24F,31, and 35B) were combined with excipients, sterile filtered and addedto APA under mixing conditions. The final concentration of eachmonovalent conjugate vaccine was 4 □g/mL (w/v PnPs) with 20 mMHistidine, 150 mM NaCl, 0.2% (w/v) PS-20 and 0.250 mg/mL (w/v Al) in theform of APA.

Example 7: Monovalent Conjugate New Zealand White Rabbit ImmunogenicityStudy (15A, 16F, 17F, 23A, 23B, 24F, 31, and 35B)

The immunogenicity of the monovalent conjugates was evaluated in a NewZealand White Rabbit (NZWR) model. Adult New Zealand White rabbits(NZWR, n=3/group) were intramuscularly (IM) immunized with 0.25 ml ofrespective monovalent conjugate vaccine on day 0 and day 14 (alternatingsides). Monovalent pneumococcal vaccine was dosed at 1 μg PnPs (15A,16F, 17F, 23A, 23B, 24F, 31, or 35B, each conjugated to CRM197) with62.5 μg aluminum phosphate adjuvant (APA) per immunization. Sera werecollected prior to study start (pre-immune) and on days 14 (post-dose 1,PD1) and 28 (post-dose 2, PD2). NZWRs were observed at least daily bytrained animal care staff for any signs of illness or distress. Thevaccine formulations in NZWRs were deemed to be safe and well tolerated.All animal experiments were performed in strict accordance with therecommendations in the Guide for Care and Use of Laboratory Animals ofthe National Institutes of Health. The NZWR experimental protocol wasapproved by the Institutional Animal Care and Use Committees at bothMerck & Co., Inc (Kenilworth, N.J.) and Covance (Denver, Pa.).

NZWR sera were tested in ELISA assays to evaluate IgG immunogenicityusing a 1-2 mg/ml respective PnPs coating concentration. Functionalantibody was determined through opsonophagocytosis assays (OPA) based onpreviously described protocols. See, e.g., Caro-Aguilar et al., 2017,Vaccine 35:865-72 and Burton et al., 2006, Clin Vaccine Immunol13(9):1004-9.

All monovalent pneumococcal conjugate vaccines were found to beimmunogenic in rabbits and generate functional antibody which killed therespective bacterial strain (data not shown). Serotype 12F was found tobe immunogenic in mice (data not shown).

1-12. (canceled)
 13. A method for preparing a polysaccharide-proteinconjugate, the method comprising reacting a polysaccharide within afirst solution with a protein within a second solution to form a thirdsolution in which a polysaccharide-protein conjugation reaction takesplace to form the polysaccharide protein conjugate, wherein the thirdsolution comprises at least 1 mM salt.
 14. The method of claim 13,wherein the salt comprises a monovalent or divalent cation.
 15. Themethod of claim 14, wherein the salt is a sodium salt, potassium salt,lithium salt, magnesium salt or calcium salt.
 16. The method of claim15, wherein the salt is sodium chloride.
 17. The method of claim 16,wherein the concentration of sodium chloride in the third solution isfrom 1 to 100 mM.
 18. The method of claim 13, wherein the salt ispresent in the first solution.
 19. The method of claim 13, wherein thesalt is present in the second solution comprising the protein.
 20. Themethod of claim 13, wherein the first solution and second solution arethe same.
 21. The method of claim 13, wherein the salt is present in thethird solution.
 22. The method of claim 13, wherein the third solutionis an aqueous solution.
 23. The method of claim 13, wherein the thirdsolution contains an aprotic solvent.
 24. The method of claim 23,wherein the aprotic solvent is DMSO.
 25. The method of claim 13, whereinthe conjugation reaction is a Schiff base reduction or reductiveamination.
 26. The method of claim 13, wherein the polysaccharide isfrom S. pneumoniae.
 27. The method of claim 26, wherein thepolysaccharide is from S. pneumoniae serotype 15A, 16F, 17F, 20, 23A,24F or 35B.
 28. The method of claim 13, wherein the protein is tetanustoxoid, diphtheria toxoid, or CRM197.
 29. The method of claim 28,wherein the protein is CRM197. 30-47. (canceled)